Mismatch repair (MMR) safeguards the integrity of the genome by recognizing and correcting errors that arise from misincorporation by the DNA polymerase or from certain chemical damages. It also prevents recombination between partially divergent DNAs that could result in genetic instability. MMR has also been implicated in the apoptotic response to DNA damage. The importance of MMR is illustrated by the finding that defects in certain MMR genes lead to a predisposition to cancer. The yeast S. cerevisiae and humans encode three MMR genes that are homologous to the E. coli MutS protein, MSH2, MSH3 and MSH6. Three yeast homologs of E. coli MutL are required for MMR, MLH1, PMS1 (PMS2 in humans) and MLH3. In humans, mutations in any of five mismatch repair gene homologs; MSH2, MLH1, MSH6, PMS2 and PMS1 contribute to both hereditary and spontaneous colon cancer. These factors participate in the recognition of mispairs, a critical step in the MMR reaction. Recently, it has been demonstrated that the mismatch recognition complexes MSH2p-MSH6p and MSH2p-MSH3p associate with the proliferating cell nuclear antigen (PCNA), a protein that plays a central role on DNA replication as a sliding clamp that enhances the processivity of DNA polymerases. The objective of this proposal is to study the consequences of the interaction between mismatch repair recognition complexes and replication proteins. We propose that these interactions increase the efficiency and fidelity of mismatch repair and may regulate the progression of replication forks when mismatches occur. We will 1) determine the multiple interactions that can form between mismatch repair and replication factors, and the consequences of these interactions in their mispair recognition activities and DNA synthesis activities, respectively. 2) Using a proteomics approach we will determine the protein composition of mispair recognition complexes that form in defined synthetic mispair substrates in vitro, and 3) Using a number of MSH2p-MSH6p and PCNA mutant proteins we will investigate if the interaction between mismatch repair proteins with PCNA allows the efficient discrimination of the DNA strands.